In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding ...In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and compliance.Pharmaceutical specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered appropriately.Conformance of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance criteria.Quality attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the specifications.Acceptance criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric specifications.Discussed in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial monographs.Outlooks are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.展开更多
Vaccination against Coronavirus disease-19(COVID-19)was pivotal to limit spread,morbidity and mortality.Our aim is to find out whether vaccines against COVID-19 lead to an immunological response stimulating the produc...Vaccination against Coronavirus disease-19(COVID-19)was pivotal to limit spread,morbidity and mortality.Our aim is to find out whether vaccines against COVID-19 lead to an immunological response stimulating the production of de novo donor specific antibodies(DSAs)or increase in mean fluorescence intensity(MFI)of pre-existing DSAs in kidney transplant recipients(KTRs).This study involved a detailed literature search through December 2nd,2023 using PubMed as the primary database.The search strategy incorporated a combination of relevant Medical Subject Headings terms and keywords:"COVID-19","SARS-CoV-2 Vaccination","Kidney,Renal Transplant",and"Donor specific antibodies".The results from related studies were collated and analyzed.A total of 6 studies were identified,encompassing 460 KTRs vaccinated against COVID-19.Immunological responses were detected in 8 KTRs of which 5 had increased MFIs,1 had de novo DSA,and 2 were categorized as either having de novo DSA or increased MFI.There were 48 KTRs with pre-existing DSAs prior to vaccination,but one study(Massa et al)did not report whether pre-existing DSAs were associated with post vaccination outcomes.Of the remaining 5 studies,35 KTRs with pre-existing DSAs were identified of which 7 KTRs(20%)developed de novo DSAs or increased MFIs.Overall,no immunological response was detected in 452(98.3%)KTRs.Our study affirms prior reports that COVID-19 vaccination is safe for KTRs,especially if there are no pre-existing DSAs.However,if KTRs have pre-existing DSAs,then an increased immunological risk may be present.These findings need to be taken cautiously as they are based on a limited number of patients so further studies are still needed for confirmation.展开更多
Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a sui...Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.展开更多
Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with ...Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL^-1and an I50 value of 0.182 μg mL^-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 μg mL^-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semiquantitation determination of the above former five organophosphorus pesticides.展开更多
Positive nucleic acid(NA)results have been found in recovered and discharged COVID-19 patients,but the proportion is unclear.This study was designed to analyze the recurrent positive rate of NA results after consecuti...Positive nucleic acid(NA)results have been found in recovered and discharged COVID-19 patients,but the proportion is unclear.This study was designed to analyze the recurrent positive rate of NA results after consecutively negative results,and the relationship between the specific antibody production and positive NA rate.According to Strengthening the Reporting of Observational Studies in Epidemiology guidelines,data of inpatients in Sino-French New City Branch of Tongji Hospital between Jan.28 and Mar.6,2020 were collected.A total of 564 COVID-19 patients over 14 years old who received the examinations of NA and antibodies against SARS-CoV-2 were included.Days of viral shedding and specific antibodies were recorded and assessed.Among NA tests in respiratory samples(throat swabs,nasopharyngeal swabs,sputum and flushing fluid in alveoli),the patients with all-negative NA results accounted for 17.20%,those with single-positive results for 46.63%,and those with multiple-positive results for 36.17%respectively.Besides,the recurrent positive NA results after consecutively negative results appeared in 66 patients(11.70%).For multiple-positive patients,median viral shedding duration was 20 days(range:1 to 57 days).Of the 205 patients who received 2 or more antibody tests,141(68.78%)had decreased IgG and IgM concentrations.IgM decreased to normal range in 24 patients,with a median of 44 days from symptom onset.Viral shedding duration was not significantly correlated with gender,age,disease severity,changes in pulmonary imaging,and antibody concentration.It is concluded that antibody level and antibody change had no significant correlation with the positive rate of NA tests and the conversion rate after continuous negative NA tests.In order to reduce the recurrent positive proportion after discharge,3 or more consecutive negative NA test results with test interval more than 24 h every time are suggested for the discharge or release from quarantine.展开更多
Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). The recombinant VHHs have a high p...Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F(ab')z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic (anti-ld) responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay (ELISA). Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.展开更多
Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated w...Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).展开更多
Allo-antibodies, particularly when donor specific, are one of the most important factors that cause both early and late graft dysfunction. The authors review the current state of the art concerning this important issu...Allo-antibodies, particularly when donor specific, are one of the most important factors that cause both early and late graft dysfunction. The authors review the current state of the art concerning this important issue in renal transplantation. Many antibodies have been recognized as mediators of renal injury. In particular donorspecific-Human Leukocyte Antigens antibodies appear to play a major role. New techniques, such as solid phase techniques and Luminex, have revealed these antibodies from patient sera. Other new techniques have uncovered alloantibodies and signs of complement activation in renal biopsy specimens. It has been acknowledged that the old concept of chronic renal injury caused by calcineurine inhibitors toxicity should be replaced in many cases by alloantibodies acting against the graft. In addition, the number of patients on waiting lists with preformed anti-human leukocyte antigens(HLA) antibodies is increasing, primarily from patients with a history of renal transplant failure already been sensitized. We should distinguish early and late acute antibody-mediated rejection from chronic antibody-mediated rejection. The latter often manifets late during the course of the posttransplant period and may be difficult to recognize if specific techniques are not applied. Different therapeutic strategies are used to control antibody-induced damage.These strategies may be applied prior to transplantation or, in the case of acute antibody-mediated rejection, after transplantation. Many new drugs are appearing at the horizon; however, these drugs are far from the clinic because they are in phase Ⅰ-Ⅱ of clinical trials. Thus the pipeline for the near future appears almost empty.展开更多
Current methods for single nucleotide polymorphism (SNP) analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification (MASA) method fo...Current methods for single nucleotide polymorphism (SNP) analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification (MASA) method for rapid SNP analysis. The method is a marriage of two technologies: MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.展开更多
The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in ...The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.展开更多
AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA excl...AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA exclusively antiH LA-Cw with 23 patients with preformed DSA antiH LA-A and/or B.RESULTS One year after transplantation there were no differencesin terms of acute rejection between the two groups(3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eG FR was not significantly different between groups(median 59 mL /min in DSA-Cw group, compared to median 51 mL /min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years(100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection(AMR) incidence was DSA strength(HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively(Log-rank P = 0.005).CONCLUSION Our data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with "classical" class I DSA.展开更多
A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemiluminescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly...A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemiluminescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy)3 ^2+(TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.展开更多
We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treate...We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.展开更多
<p align="justify"> <span style="font-family:Verdana;"></span><span style="font-family:Verdana;"></span>Myeloproliferative neoplasms (MPNs) are a group of cl...<p align="justify"> <span style="font-family:Verdana;"></span><span style="font-family:Verdana;"></span>Myeloproliferative neoplasms (MPNs) are a group of clonal haematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. According to WHO classification, the Janus associated kinase 2 (<em>JAK</em>2) V617F mutation is one of the major diagnostic criteria in BCR-ABL1 negative myeloproliferative neoplasms. The aim of this study is to detect the <em>JAK</em>2 (V617F) mutation in patients with myeloproliferative neoplasms to get accurate diagnosis and proper management. A total of 90 clinically diagnosed MPN patients attending to Department of Clinical Haematology, Yangon General Hospital were enrolled in this study. The mean age was 53.4 ± 14 years which ranged from 16 to 81 years old and male and female ratio was 2.4:1. The identification of <em>JAK</em>2 (V617F) point mutation was found to be positive in 44/90 MPN patients (48.9%). According to MPN subtypes, the <em>JAK</em>2 mutation positivity was found in 19 out of 46 polycythemia vera patients (41.3%), 17 out of 25 essential thrombocythemia patients (68%), 8 out of 15 primary myelofibrosis patients (53.3%), 0 of 4 others myeloproliferative neoplasms (0%). Confirmation of each of nine <em>JAK</em>2 mutation positive and negative samples was done by Sanger sequencing. The arterial or venous thrombotic attack was found in 32/44 <em>JAK</em>2 mutation positive cases (72.7%) and 12/44 <em>JAK</em>2 mutation negative cases (27.3%). The association between thrombotic attack and presence of <em>JAK</em>2 mutation was statistically significance with p = 0.000. The diagnosis of myeloproliferative neoplasms mainly relies on the molecular genetics according to WHO classification. The Allele specific PCR reaction is sensitive, simple test and relatively cost-effective. Therefore, the identification of <em>JAK</em>2 (V617F) somatic point mutation by AS-PCR should be implemented as a routine diagnosis procedure for patients with chronic and suspected myeloproliferative neoplasms. </p>展开更多
using enzymellnked lmmunosorheut assay (ELlsA), we had determined the speclrlc antibodies of IgM and lgA to CMV In 14 patients with BAT, 36 marrow donors and 682 blood donorsfrom 1991 to 1996. The antibodies detected ...using enzymellnked lmmunosorheut assay (ELlsA), we had determined the speclrlc antibodies of IgM and lgA to CMV In 14 patients with BAT, 36 marrow donors and 682 blood donorsfrom 1991 to 1996. The antibodies detected were negative in 14 patients, 16. 16% POsitive in marrowdonors and 34. 31 % in blood donors respectively. These resultS suggested that there was a higher active or recent CMV Inrectlou in blood donors in XI'an area. In order to prevent transfusion-acquiredCMV infectlony it is nessessary ror us to screen out negative CMV antibodies donors in BAT, whichhas great value for clinical application.展开更多
Targeted treatment of cancer with monoclonal antibodies increases the benefit for patients. In order to improve the anti-tumor activity of monoclonal antibodies, multi-specific antibodies have entered the research fie...Targeted treatment of cancer with monoclonal antibodies increases the benefit for patients. In order to improve the anti-tumor activity of monoclonal antibodies, multi-specific antibodies have entered the research field. The emergence of various techniques to produce multi-specific recombinant antibody molecules has led to the selection of target combinations in various forms. To date, only a few multi-specific constructs have entered phase III clinical trials, in contrast to classical monoclonal antibodies. Some of the format options are outlined from a technical point of view. We focus on the achievements and prospects of the underlying technologies for generating biand multispecific antibodies.展开更多
To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rab...To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents.展开更多
AIM:To study the diagnosis of Helicobacter pylori(H pylori) infection through the determination of serum levels of anti- H pylori IgG and IgA antibodies,and the levels of anti-H pylori IgA antibodies in duodenal fluid...AIM:To study the diagnosis of Helicobacter pylori(H pylori) infection through the determination of serum levels of anti- H pylori IgG and IgA antibodies,and the levels of anti-H pylori IgA antibodies in duodenal fluid. METHODS:Data were collected from 93 patients submitted to upper digestive endoscopy due to dyspeptic symptoms. The patients were either negative(group A)or positive (group B)to H pylori by means of both histological detection and urease tests.Before endoscopy,peripheral blood was collected for the investigation of anti-H pylori IgG and IgA antibodies.To perform the urease test,biopsies were obtained from the gastric antrum.For the histological evaluation,biopsies were collected from the gastric antrum (greater and lesser curvatures)and the gastric body. Following this,duodenal fluid was collected from the first and second portions of the duodenum.For the serological assaying of anti-Hpylori IgG and IgA,and anti-Hpylori IgA in duodenal fluids,the ELISA method was utilized. RESULTS:The concentration of serum IgG showed sensitivity of 64.0%,specificity of 83.7%,positive predictive value of 82.0%,negative predictive value of 66.6% and accuracy of 73.1% for the diagnosis of H pylori infection.For the same purpose,serum IgA showed sensitivity of 72.0%, specificity of 65.9%,positive predictive value of 72.0%, negative predictive value of 67.4% and accuracy of 69.8%. If the serological tests were considered together,i.e.when both were positive or negative,the accuracy was 80.0%, sensitivity was 86.6%,specificity was 74.2%,positive predictive value was 74.2% and negative predictive value was 86.6%.When values obtained in the test for detecting IgA in the duodenal fluid were analyzed,no significant difference(P=0.43)was observed between the values obtained from patients with or without H pylori infection. CONCLUSION:The results of serum IgG and IgA tests for H pylori detection when used simultaneously,are more efficient in accuracy,sensitivity and negative predictive value, than those when used alone.The concentration of IgA antibodies in duodenal fluid is not useful in identifying patients with or without H pylori.展开更多
Testis-specific protease 50(TSP50) has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential targ...Testis-specific protease 50(TSP50) has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies(mAbs) with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all of the 9 different clones can specifically bind to TSP50.The mAbs against TSP50 we generated could be good tools for both basic and clinical studies.展开更多
基金supported by the Grant for Development of New Faculty Staff,Ratchadaphiseksomphot Endowment Fund,Chula-longkorn University,Thailand(Grant No.:DNS64_047_33_003_1 to Patanachai K.Limpikirati)Grant for Development of New Scholar,Office of the Permanent Secretary,Ministry of Higher Ed-ucation,Science,Research and Innovation,Thailand(Grant No.:RGNS64_012 to Patanachai K.Limpikirati).
文摘In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and compliance.Pharmaceutical specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered appropriately.Conformance of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance criteria.Quality attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the specifications.Acceptance criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric specifications.Discussed in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial monographs.Outlooks are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.
文摘Vaccination against Coronavirus disease-19(COVID-19)was pivotal to limit spread,morbidity and mortality.Our aim is to find out whether vaccines against COVID-19 lead to an immunological response stimulating the production of de novo donor specific antibodies(DSAs)or increase in mean fluorescence intensity(MFI)of pre-existing DSAs in kidney transplant recipients(KTRs).This study involved a detailed literature search through December 2nd,2023 using PubMed as the primary database.The search strategy incorporated a combination of relevant Medical Subject Headings terms and keywords:"COVID-19","SARS-CoV-2 Vaccination","Kidney,Renal Transplant",and"Donor specific antibodies".The results from related studies were collated and analyzed.A total of 6 studies were identified,encompassing 460 KTRs vaccinated against COVID-19.Immunological responses were detected in 8 KTRs of which 5 had increased MFIs,1 had de novo DSA,and 2 were categorized as either having de novo DSA or increased MFI.There were 48 KTRs with pre-existing DSAs prior to vaccination,but one study(Massa et al)did not report whether pre-existing DSAs were associated with post vaccination outcomes.Of the remaining 5 studies,35 KTRs with pre-existing DSAs were identified of which 7 KTRs(20%)developed de novo DSAs or increased MFIs.Overall,no immunological response was detected in 452(98.3%)KTRs.Our study affirms prior reports that COVID-19 vaccination is safe for KTRs,especially if there are no pre-existing DSAs.However,if KTRs have pre-existing DSAs,then an increased immunological risk may be present.These findings need to be taken cautiously as they are based on a limited number of patients so further studies are still needed for confirmation.
基金supported by grants from the National Natural Science Foundation of China(31572381)National Thousand Youth Talents Plan of the International Science and Technology Cooperation Project of China(2013DFA31420)Science and Technology Innovation Capability Promotion Program of the Science and Technology Department of Qinghai Province(2015-ZJ-712)
文摘Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.
基金supported by the grants of National Natural Science Foundation of China (30471155)National 863 Program of China (2006AA06Z411)
文摘Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL^-1and an I50 value of 0.182 μg mL^-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 μg mL^-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semiquantitation determination of the above former five organophosphorus pesticides.
文摘Positive nucleic acid(NA)results have been found in recovered and discharged COVID-19 patients,but the proportion is unclear.This study was designed to analyze the recurrent positive rate of NA results after consecutively negative results,and the relationship between the specific antibody production and positive NA rate.According to Strengthening the Reporting of Observational Studies in Epidemiology guidelines,data of inpatients in Sino-French New City Branch of Tongji Hospital between Jan.28 and Mar.6,2020 were collected.A total of 564 COVID-19 patients over 14 years old who received the examinations of NA and antibodies against SARS-CoV-2 were included.Days of viral shedding and specific antibodies were recorded and assessed.Among NA tests in respiratory samples(throat swabs,nasopharyngeal swabs,sputum and flushing fluid in alveoli),the patients with all-negative NA results accounted for 17.20%,those with single-positive results for 46.63%,and those with multiple-positive results for 36.17%respectively.Besides,the recurrent positive NA results after consecutively negative results appeared in 66 patients(11.70%).For multiple-positive patients,median viral shedding duration was 20 days(range:1 to 57 days).Of the 205 patients who received 2 or more antibody tests,141(68.78%)had decreased IgG and IgM concentrations.IgM decreased to normal range in 24 patients,with a median of 44 days from symptom onset.Viral shedding duration was not significantly correlated with gender,age,disease severity,changes in pulmonary imaging,and antibody concentration.It is concluded that antibody level and antibody change had no significant correlation with the positive rate of NA tests and the conversion rate after continuous negative NA tests.In order to reduce the recurrent positive proportion after discharge,3 or more consecutive negative NA test results with test interval more than 24 h every time are suggested for the discharge or release from quarantine.
基金supported by a grant from the National Basic Research Program of China(2013CB127804)the National Natural Science Funds(31171696,China)the Research Program of the State Key Laboratory of Food Science and Technology,Nanchang University(SKLF-MB-201002)
文摘Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F(ab')z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic (anti-ld) responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay (ELISA). Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.
文摘Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).
文摘Allo-antibodies, particularly when donor specific, are one of the most important factors that cause both early and late graft dysfunction. The authors review the current state of the art concerning this important issue in renal transplantation. Many antibodies have been recognized as mediators of renal injury. In particular donorspecific-Human Leukocyte Antigens antibodies appear to play a major role. New techniques, such as solid phase techniques and Luminex, have revealed these antibodies from patient sera. Other new techniques have uncovered alloantibodies and signs of complement activation in renal biopsy specimens. It has been acknowledged that the old concept of chronic renal injury caused by calcineurine inhibitors toxicity should be replaced in many cases by alloantibodies acting against the graft. In addition, the number of patients on waiting lists with preformed anti-human leukocyte antigens(HLA) antibodies is increasing, primarily from patients with a history of renal transplant failure already been sensitized. We should distinguish early and late acute antibody-mediated rejection from chronic antibody-mediated rejection. The latter often manifets late during the course of the posttransplant period and may be difficult to recognize if specific techniques are not applied. Different therapeutic strategies are used to control antibody-induced damage.These strategies may be applied prior to transplantation or, in the case of acute antibody-mediated rejection, after transplantation. Many new drugs are appearing at the horizon; however, these drugs are far from the clinic because they are in phase Ⅰ-Ⅱ of clinical trials. Thus the pipeline for the near future appears almost empty.
基金This research is supported by the National Natural Science Foundation of China(60378043,30470494)the Natural Science Foundation of Guangdong Province(015012,04010394).
文摘Current methods for single nucleotide polymorphism (SNP) analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification (MASA) method for rapid SNP analysis. The method is a marriage of two technologies: MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.
基金Financial support received from Department of Biotechnology,Government of India vide grant No.BT/PR-8953/BCE/08/533/2007project sanctioned against grant No.BT/04/NE/2009financial support from Department of Science&Technology,Government of India in the form of a research fellowship under the INSPIRE program
文摘The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.
文摘AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA exclusively antiH LA-Cw with 23 patients with preformed DSA antiH LA-A and/or B.RESULTS One year after transplantation there were no differencesin terms of acute rejection between the two groups(3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eG FR was not significantly different between groups(median 59 mL /min in DSA-Cw group, compared to median 51 mL /min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years(100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection(AMR) incidence was DSA strength(HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively(Log-rank P = 0.005).CONCLUSION Our data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with "classical" class I DSA.
基金the National Natural Science Foundation of China (Nos. 30600128, 30670507,30470494) the Natural Science Foundation of Guangdong Province (No. 015012).
文摘A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemiluminescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy)3 ^2+(TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA-ECL assay. The method is useful in SNP analysis due to its sensitivity,safety, and simplicity.
文摘We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.
文摘<p align="justify"> <span style="font-family:Verdana;"></span><span style="font-family:Verdana;"></span>Myeloproliferative neoplasms (MPNs) are a group of clonal haematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. According to WHO classification, the Janus associated kinase 2 (<em>JAK</em>2) V617F mutation is one of the major diagnostic criteria in BCR-ABL1 negative myeloproliferative neoplasms. The aim of this study is to detect the <em>JAK</em>2 (V617F) mutation in patients with myeloproliferative neoplasms to get accurate diagnosis and proper management. A total of 90 clinically diagnosed MPN patients attending to Department of Clinical Haematology, Yangon General Hospital were enrolled in this study. The mean age was 53.4 ± 14 years which ranged from 16 to 81 years old and male and female ratio was 2.4:1. The identification of <em>JAK</em>2 (V617F) point mutation was found to be positive in 44/90 MPN patients (48.9%). According to MPN subtypes, the <em>JAK</em>2 mutation positivity was found in 19 out of 46 polycythemia vera patients (41.3%), 17 out of 25 essential thrombocythemia patients (68%), 8 out of 15 primary myelofibrosis patients (53.3%), 0 of 4 others myeloproliferative neoplasms (0%). Confirmation of each of nine <em>JAK</em>2 mutation positive and negative samples was done by Sanger sequencing. The arterial or venous thrombotic attack was found in 32/44 <em>JAK</em>2 mutation positive cases (72.7%) and 12/44 <em>JAK</em>2 mutation negative cases (27.3%). The association between thrombotic attack and presence of <em>JAK</em>2 mutation was statistically significance with p = 0.000. The diagnosis of myeloproliferative neoplasms mainly relies on the molecular genetics according to WHO classification. The Allele specific PCR reaction is sensitive, simple test and relatively cost-effective. Therefore, the identification of <em>JAK</em>2 (V617F) somatic point mutation by AS-PCR should be implemented as a routine diagnosis procedure for patients with chronic and suspected myeloproliferative neoplasms. </p>
文摘using enzymellnked lmmunosorheut assay (ELlsA), we had determined the speclrlc antibodies of IgM and lgA to CMV In 14 patients with BAT, 36 marrow donors and 682 blood donorsfrom 1991 to 1996. The antibodies detected were negative in 14 patients, 16. 16% POsitive in marrowdonors and 34. 31 % in blood donors respectively. These resultS suggested that there was a higher active or recent CMV Inrectlou in blood donors in XI'an area. In order to prevent transfusion-acquiredCMV infectlony it is nessessary ror us to screen out negative CMV antibodies donors in BAT, whichhas great value for clinical application.
文摘Targeted treatment of cancer with monoclonal antibodies increases the benefit for patients. In order to improve the anti-tumor activity of monoclonal antibodies, multi-specific antibodies have entered the research field. The emergence of various techniques to produce multi-specific recombinant antibody molecules has led to the selection of target combinations in various forms. To date, only a few multi-specific constructs have entered phase III clinical trials, in contrast to classical monoclonal antibodies. Some of the format options are outlined from a technical point of view. We focus on the achievements and prospects of the underlying technologies for generating biand multispecific antibodies.
基金National Department Public Benefit Research Foundation (201103032)Pathogens Network Monitoring Technology Research (2008ZX10004-008)
文摘To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma cells were fused according to conventional methods: the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000, respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific anti-rabies virus monoclonal antibodies, which makes them well suited for the development of rabies diagnosis reagents.
文摘AIM:To study the diagnosis of Helicobacter pylori(H pylori) infection through the determination of serum levels of anti- H pylori IgG and IgA antibodies,and the levels of anti-H pylori IgA antibodies in duodenal fluid. METHODS:Data were collected from 93 patients submitted to upper digestive endoscopy due to dyspeptic symptoms. The patients were either negative(group A)or positive (group B)to H pylori by means of both histological detection and urease tests.Before endoscopy,peripheral blood was collected for the investigation of anti-H pylori IgG and IgA antibodies.To perform the urease test,biopsies were obtained from the gastric antrum.For the histological evaluation,biopsies were collected from the gastric antrum (greater and lesser curvatures)and the gastric body. Following this,duodenal fluid was collected from the first and second portions of the duodenum.For the serological assaying of anti-Hpylori IgG and IgA,and anti-Hpylori IgA in duodenal fluids,the ELISA method was utilized. RESULTS:The concentration of serum IgG showed sensitivity of 64.0%,specificity of 83.7%,positive predictive value of 82.0%,negative predictive value of 66.6% and accuracy of 73.1% for the diagnosis of H pylori infection.For the same purpose,serum IgA showed sensitivity of 72.0%, specificity of 65.9%,positive predictive value of 72.0%, negative predictive value of 67.4% and accuracy of 69.8%. If the serological tests were considered together,i.e.when both were positive or negative,the accuracy was 80.0%, sensitivity was 86.6%,specificity was 74.2%,positive predictive value was 74.2% and negative predictive value was 86.6%.When values obtained in the test for detecting IgA in the duodenal fluid were analyzed,no significant difference(P=0.43)was observed between the values obtained from patients with or without H pylori infection. CONCLUSION:The results of serum IgG and IgA tests for H pylori detection when used simultaneously,are more efficient in accuracy,sensitivity and negative predictive value, than those when used alone.The concentration of IgA antibodies in duodenal fluid is not useful in identifying patients with or without H pylori.
基金Supported by the National Natural Science Foundation of China(No.30672068)the Distinguished Young Scholars Fund of Jilin Province,China(No.20050114)+1 种基金the Key Grant of Jilin Province Science & Technology Committee,China(Nos.20060923-01,200805131)the Key Grant of Changchun City Science & Technology Committee,China(No.06GG147)
文摘Testis-specific protease 50(TSP50) has been identified as a testis-specific protein that is expressed abnormally in most human breast cancer samples,which makes it an attractive molecular marker and a potential target for diagnosis and therapy.In the present study,we prepared a panel of monoclonal antibodies(mAbs) with high specificity and sensitivity against TSP50 by hybridoma method and characterized them by ELISA,Western blot,immunofluroescence and immunohistochemical analyses.The results show that all of the 9 different clones can specifically bind to TSP50.The mAbs against TSP50 we generated could be good tools for both basic and clinical studies.